Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by the accumulation of insoluble amyloid deposits of the amyloid-β (Aβ) protein generated by the cleavage of the amyloid precursor protein (APP) (Blennow, K., et al., Lancet 368: 387-403 (2006)). AD is one of several diseases caused by protein misfolding, which includes over 22 other known ailments such as Parkinson's disease and Type II diabetes (Dobson, C. M., Protein Pept. Lett. 13: 219-227 (2006)). There are at present no effective treatments against AD and most of the other protein misfolding diseases, partly owing to the fact that it is still unclear if and how these deposits are toxic. In fact, focus has recently shifted away from the insoluble amyloid deposits, which previously were believed to be the cause of AD, towards soluble Aβ oligomeric species as the toxic agent (Haass, C. and Selkoe D. J., Nature Reviews Mol. Cell. Biol. 8: 101-112 (2007)). In addition, conformation-dependent antibodies raised against Aβ oligomers (e.g. the polyclonal A11 antibody) have shown to be reactive towards other oligomeric species in addition to Aβ, thus suggesting that these toxic structures are generic in the protein misfolding diseases although different proteins are involved (Glabe, C. G., Trends Biochem. Sci. 29: 542-547 (2004)). Much effort is therefore currently being focused worldwide on the isolation and characterization of such soluble oligomers for drug-screening and immunogenic purposes. However, both monomeric and oligomeric Aβ proteins have a high tendency to aggregate further into fibrils. This fibrillation is a spontaneous nucleation-dependent polymerization reaction for which the rate is sensitive to peptide concentration (Lomakin, A., et al., Proc. Natl. Acad. Sci. USA 93: 1125-1129 (1996)). In consequence, fibrillogenesis seriously limits the longevity of the oligomeric preparations, and also the concentrations at which they can be kept.
The wild-type Aβ protein implicated in AD is a very fibrillation prone peptide at concentrations>100 μM. At lower concentrations (>20 μM to <100 μM) said protein has a tendency to slowly oligomerize (oligomers are soluble structures containing a plurality of monomers) prior to proceeding into the inert fibrillar state. This oligomeric state has recently been implicated as a neurotoxic agent and, therefore, as the toxic species involved in AD (Haass, C., and Selkoe, D. J., Nature Reviews Mol. Cell. Biol. 8: 101-112 (2007)). The prevalent method for producing these toxic structures at physiological conditions involve incubating peptide solutions at 20-100 μM and 4° C. in cell culture medium (F-12) or in buffered salt solutions for several days (Lambert, M. P., et al., Proc. Natl. Acad. Sci. USA. 95: 6448-6453 (1998); Stine, W. B., et al., J. Biol. Chem. 278: 11612-11622 (2003)). Because nucleation and elongation rates are strongly dependent on peptide concentration (Lomakin, A., et al., Proc. Natl. Acad. Sci. USA 93: 1125-1129 (1996)), any increase in the concentration of peptide above 100 μM will be detrimental to the stability of the oligomer preparation. In the classical view of amyloid fibril assembly, the nuclei triggering polymerization and the oligomeric structures are even believed to be the same species. Oligomer preparations therefore have a very limited and unpredictable (aggregation nucleation is a spontaneous event) lifespan once they are formed. In dilute solutions (20 μM to 25 μM) at 4° C. oligomer preparations of Aβ(42) are typically stable only for 24 h, and those of Aβ(40) for maybe a week. In less dilute solutions, >100 μM, the insolubility of the Aβ protein decreases dramatically. The fibrillation process is thus a serious drawback when, as in screening assays for medicaments, stable proteins are required at relatively high concentration.
The aim of the present invention is to overcome these problems by providing engineered Aβ peptides that form stabile oligomers.
There are no previous reports of stabilized Aβ hairpin structures and no reports of Aβ peptides containing only the A21C/A30C disulphide without additional complicating cysteines. US 2006/0018918 discloses Aβ isomers based on a consideration of the Aβ primary structure alone with multiple cysteine replacement of all Ser and Ala in general with the intent of stabilizing non-native conformations of Aβ to be used as vaccines. The A21C and A30C mutations are obtained together with three or four additional cysteine mutants at position Ala2, Ser8, Ser26, and Ala42. Oxidation of these Aβ isomers produces a mixture of 15 possible isoforms with different intramolecular disulphide bonds where only three of the 15 possible isoforms will contain an A21C/A30C disulphide bond, but always in combination with additional disulphide bonds.
Previous Aβ peptide disulfide mutants reported in the literature are L17C/L34C, L17C/M35C, and L17C/V36C (Shivaprasad, S, and Wetzel, R., Biochemistry 43: 153-15317 (2004)). These mutations are all non-conservative replacements. Furthermore, these mutants were made specifically to investigate the proximity of Leu17 to Leu34, Met35, and Val36 in the fibril structure. Shivaprasad and Wetzel present data that demonstrate that these three mutants undergo fibrillogenesis with lag times that are nearly identical for the reduced (with the mutated residues as cysteines) and oxidized (with the mutated residues as cystines) mutants. Other disulphide mutants, which are also non-conservative replacements, have been published by the same authors, namely V18C/L34C, F19C/A30C, F19C/I32C, and F19C/L34C (Wetzel, R., et al., Biochemistry 46: 1-10 (2007)). These mutants were found to behave similarly to the L17C/L34C, L17C/M35C, and L17C/V36C mutants. Hence, all previously published disulphide mutants of the Aβ protein have been demonstrated to readily aggregate into fibrils of at least similar stability to the fibrils obtained from wild-type peptide. These oxidized derivatives of the L17C/L34C, L17C/M35C, L17C/V36C, V18C/L34C, F19C/A30C, F19C/I32C, and F19C/L34C mutants all fibrillate because they are incompatible with the hairpin structure presented in Hoyer et al. (Hoyer, W. et al., Proc. Natl. Acad. Sci. USA. 105: 5099-5104 (2008)) whereas they are compatible with current models of Aβ-peptide fibril structures where the two β-strands pack against each other (see e.g. Petkova, A. T., et al., Biochemistry 45: 498-512).
Aβ oligomer preparations are described in WO 2007/005358, WO 2007/005359, WO 2007/142320 and WO 2004/067561. These described oligomer preparations have been obtained by using intermolecular crosslinkers (WO 2007/005358), non-physiological pH (pH 9 in WO 2007/005359) and/or additives (40% glycerol or TFE were claimed to stabilize oligomers at 37° C. in WO 2007/005359; GM1 ganglioside in WO 2007/142320; and SDS in WO 2004/067561).